Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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High trait impulsivity predicts food addiction-like behavior in the rat

Clara Velázquez-Sánchez, Ph.D.a; Antonio Ferragud, M.S.

a; Catherine F Moore, B.A.

a; Barry

J Everitt, Sc.D.b; Valentina Sabino, Ph.D.

a; Pietro Cottone, Ph.D.

a,*

aLaboratory of Addictive Disorders, Department of Pharmacology and Experimental

Therapeutics and Department of Psychiatry, Boston University School of Medicine, 72 E

Concord Street, Boston, MA 02118, USA

bBehavioral and Clinical Neuroscience Institute and Department of Experimental

Psychology, University of Cambridge, Downing Street, Cambridge, Cambridgeshire, CB2

3EB, UK

Running title: High impulsivity predicts food addiction

* Correspondence to: Pietro Cottone, Laboratory of Addictive Disorders, Departments of

Pharmacology and Psychiatry, Boston University School of Medicine, 72 E Concord St,

Boston, MA 02118, USA. Tel: +1 617-638-5662, E-mail: cottone@bu.edu

Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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SUPPLEMENTARY MATERIALS AND METHODS

Subjects

Male Wistar rats (n=47), 45 days old on arrival (Charles River, Wilmington, MA),

were triple-housed in wire-topped plastic cages in a 12-h reverse light cycle (lights off at

11:00 am) AAALAC-approved humidity-(60%) and temperature-controlled (22°C) vivarium.

Animals were allowed a four-day habituation period to the research facility. Corn-based

chow (Harlan Teklad LM-485 Diet 7012) and water were available in the home cage.

Procedures adhered to the National Institutes of Health Guide for the Care and Use of

Laboratory Animals and the Principles of Laboratory Animal Care, and were approved by

Boston University Medical Campus Institutional Animal Care and Use Committee. No

experimental procedures involved food or water restriction/deprivation.

Apparatus

Operant chambers (30×24×29 cm) were identical to those previously described (1, 2).

Briefly, the chambers (Med Associates, St Albans, VT) had wire- mesh floors and were

located in ventilated, sound-attenuating enclosures (66×56×36 cm). A syringe pump

delivered the solution into a stainless steel receptacle located 2 cm above the floor in the

middle of the left wall of the chamber. Levers were placed 3.2 cm next to the drinking cup.

Food pellets were delivered by a dispenser into a nosepoke aperture on the opposite wall of

the chamber. 28-V stimulus cue-lights were located above each lever and above the food

magazine. Water was also delivered by a solenoid into a liquid cup nosepoke receptacle

placed in the center of the right wall. All responses were recorded automatically by a

microcomputer with 10 ms resolution.

Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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Differential reinforcement of low rates of responding (DRL) task in ad libitum fed rats

Performance on the DRL task has been used to provide a measure of impulsive action,

defined as the inability to withhold a response (3-5). The DRL procedure was adapted from

previous reports (6-8) in ad libitum fed rats. Rats were trained to lever press for a

“supersaccharin” solution in an overnight fixed ratio 1 (FR1) self-administration session (9,

10), consisting of 1.5% w/v glucose and 0.4% w/v saccharin (9, 10). Rats were then trained

on a DRL-5 s schedule for four sessions, during which a lever press resulted in the delivery

of the supersaccharin solution if at least 5 s had elapsed since the previous lever press. If the

rat made a premature lever press, the 5 s time period was reset; thus, rats were only

reinforced if they withheld a response for longer than 5 s. Rats were then trained for four

sessions on a DRL-10 s schedule, during which the response had to be withheld for 10 s in

order to obtain reinforcement. Finally, rats were given 15 sessions on a DRL-15 s schedule.

Impulsive action was assessed on the last four sessions of the DRL-15 s schedule, and was

defined as efficiency [ratio between the rewarded responses and the total (rewarded +

incorrect) responses], with higher efficiency reflecting more accurate performance, indicative

of less impulsive action. During the last four sessions of DRL-15 s schedule, responding was

highly stable and an intraclass correlation analysis of the efficiency showed very high internal

consistency (Intraclass Correlation, Efficiency: ICC[2,4]=0.93, F(28,84)=15.61 p<0.0001)

across the 4 days. The high intraclass correlation coefficient suggests very stable individual

differences. Rats were ranked according to their efficiency scores and subjects falling above

the 60th

percentile were assigned to the Low-impulsive group, while subjects falling below the

40th

percentile were assigned to the High-impulsive group.

Spontaneous locomotor activity

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The analysis of locomotor reactivity to a novel environment was carried out during

the first two hours of the dark phase as previously shown (11). The motor activity was

measured in novel Plexiglas chambers (27×48×20 cm) using an Opto –M3 activity system

(Columbus Instruments, Columbus, OH). The Opto-M3 system consisted of a series of 16

sensor beams spaced 2.54 cm apart and able to measure horizontal activity. Sensor beams

were located along the longest side of the horizontal plane of the cage. White noise was

present throughout testing. Rats were ranked according to their photocell beam breaks and

subjects falling below the 40th

percentile were assigned to the Low-responder group, while

subjects falling above the 60th

percentile were assigned to the High-responder group.

Elevated Plus-Maze test

The elevated plus-maze (12-14) apparatus was made of black Plexiglas and consisted

of four arms (50 cm long 10 cm wide). Two arms had 40-cm-high dark walls (enclosed

arms), and two arms had 0.5-cm-high ledges (open arms). The maze was elevated to a height

of 50 cm. Open arms received 1.5–2.0 lux of illumination. Animals were habituated to the

anteroom the day before testing. On the day of testing, rats were kept in the quiet, dark

anteroom for at least 2 h before testing. White noise was present throughout habituation and

testing. For testing, rats were placed individually onto the center of the maze facing an open

arm and removed after a 5-min period. The apparatus was cleaned with water and dried

between subjects. The primary measures were the percentage of total arm time directed

toward the open arms [i.e., 100*open arm/(open arm+closed arm)], a validated index of

anxiety-related behavior and the number of closed arm entries, a specific index of locomotor

activity. Rats were ranked according to their percentage of open arm time and subjects falling

above the 60th

percentile were assigned to the Low-anxiety group, while subjects falling

below the 40th

percentile were assigned to the High-anxiety group.

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Binge-like eating procedure in ad libitum fed rats

Following the DRL procedure and separation of Low- and High-impulsive rats, ad

libitum fed rats were trained to acquire a nosepoke response for food and water in individual

test cages (30×24×29 cm) on a fixed ratio 1 (FR1) continuous schedule of reinforcement in 1

h sessions, as described previously (1, 2). The operant boxes had grid floors and were located

in ventilated, sound-attenuating enclosures (66×56×36 cm). Food reinforcers were delivered

by a pellet dispenser (Med Associates Inc., St. Albans, VT). During instrumental training,

food pellets were 45-mg precision pellets, identical in composition to the diet that rats

received in the home cage as ~5 g extruded pellets (5TUM diet formulated as 4-5g extruded

pellets, 65.5% [kcal] carbohydrate, 10.4% fat, 24.1% protein, metabolizable energy 330

cal/100 g; TestDiet, Richmond, IN). Therefore, in the operant chambers, rats were provided

with a diet identical to the one received in the home cage to ensure that Chow rats’ food

intake during operant sessions was not influenced by any hedonic factor, but only by

homeostatic needs (1, 2, 15, 16). Pellet delivery was paired with a light-cue located above the

nosepoke hole. Water reinforcers were 100 µl in volume, delivered by a solenoid into a liquid

cup nosepoke receptacle. The sessions were performed daily before dark cycle onset. After

attaining stable baseline performance in the 1-hr self-administration sessions, the testing

procedure was initiated. Rats were assigned to either a “Chow” control group (Low-

impulsive/Chow and High-impulsive/Chow) and continued to receive the same 45-mg chow

pellets offered in the training phase, or a “Palatable” group (Low-impulsive/Palatable, and

High-impulsive/Palatable), which instead received a nutritionally complete, chocolate-

flavored, high sucrose (50% kcal), AIN-76A-based diet, comparable in macronutrient

composition and energy density to the chow diet (chocolate-flavored Formula 5TUL: 66.7%

[kcal] carbohydrate, 12.7% fat, 20.6% protein, metabolizable energy 344 cal/100 g;

Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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formulated as 45 mg precision food pellets; TestDiet, Richmond, IN). This chocolate-

flavored diet is strongly preferred by all rats (16, 17). The rats were tested daily for 1 h

sessions. After acquiring stable responding under the FR1 schedule of reinforcement, the

number of responses required to obtain one pellet was increased from FR1 to FR3 during 4

consecutive sessions, and then from FR3 to FR5 for 4 additional sessions.

Progressive ratio schedule of reinforcement for food

Following testing under the fixed ratio schedules, rats were moved to a progressive

ratio schedule of reinforcement, where the number of responses required to obtain a food

pellet increased with successive food reinforcers based on the following shallow exponential

progression: response ratio = [4·(e# of reinforcer*0.075

) −3.8], rounded to the nearest integer. To

avoid unintended session starts (e.g., due to exploratory, rather than food-directed activity),

the first reinforcement required three responses. Thus, the progressive ratio schedule was 3,

1, 1, 2, 2, 2, 3, 3, 4, 5, 5, 6, 7, 8, 9, 9, 11, 12, 13, 14, 16, 17, 19, 20, 22, 24, 27, 29, etc.

responses. Sessions ended when subjects had not completed a ratio for 14 min, with the last

completed ratio defined as the breakpoint. This criterion was used because male Wistar rats

do not voluntarily wait longer than 14 min between meals without eating a pellet (12, 18).

Thus, sessions involved rats initiating a meal but with the meal ending prematurely (prior to

full satiation); when escalating, response requirements surpassed the rats' breakpoints. The

latency was set to a maximum of 1 h. At the end of each session, subjects were returned to

their home cage, where the regular chow was always available ad libitum.

Light/Dark conflict test

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The day after the last PR session, rats were tested in a light/dark conflict test. The

same rats used for the development of the binge-like eating procedure were tested in a

light/dark rectangular box (50×100×35 cm) in which the aversive, bright compartment

(50×70×35 cm) was illuminated by a 60 lux light. The dark compartment (50×30×35 cm) had

an opaque cover and ~0 lux of light. The two compartments were connected by an open

doorway which allowed the subjects to move freely between the two (2, 19, 20). A shallow,

metal cup containing a pre-weighed amount of the same food received during self-

administration (45-mg chow pellets for Chow rats or 45-mg chocolate pellets for Palatable

rats) was positioned in the center of the light compartment. Rats were habituated to an ante-

room 2 h prior to testing. White noise was present during both habituation and testing. On

the test day, rats were placed into the light compartment, facing both the food cup and the

doorway. Under normal, control conditions, eating behavior is typically suppressed when a

rat is in the aversive bright environment; a significant increase in food intake in spite of these

adverse conditions, as compared to control conditions, was operationalized as a construct of

“compulsive-like eating” (2, 11, 20-24). The apparatus was cleaned with a water-dampened

cloth after each subject. Rats had access to food ad libitum at all times; water was not

available during the 10-min test.

∆FosB immunohistochemistry

Perfusions and immunohistochemistry

At the end of the behavioral procedures, animals were transcardially perfused with

0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 phosphate buffer (PB) under

deep isoflurane anesthesia. Brains were removed, postfixed in 4% PFA for 24 h and then

stored in 30% sucrose in PB buffer at 4°C until saturation. Brains were sectioned coronally at

30µm using a cryostat and stored in a cryoprotectant solution at -20°C. For technical reasons

Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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related to the section quality four subjects were excluded. Every sixth section from 2.16 mm

of the nucleus accumbens and dorsal striatum were selected in a systematic manner and

processed for immunohistochemistry. Briefly, for ∆FosB immunohistochemistry, mounted

sections were washed in a phosphate saline (PBS) buffer. After the initial washing, sections

were incubated in 1% hydrogen peroxide PBS solution for 10 min to block endogenous

peroxidases. Sections were washed again and placed in blocking solution (5% normal goat

serum and 0.4% Triton X100) for 1 h respectively. Slides were transferred into primary

antibodies (∆FosB, 1:500, sc-48 Santa Cruz Biotechnology) (25, 26) in blocking solution and

incubated for 24 h at 4°C. Following an additional wash, sections were incubated in the

secondary antibody (biotinylated goat anti-rabbit 1:500 in PBS, Vector Laboratories) for 1 h

at room temperature. Sections were washed and incubated in an avidin–biotin horseradish

peroxidase ABC solution (1:1000 in PBS, Vector Laboratories) for 1 h. Slides were then

incubated using diaminobenzidine substrate kit (Vector Laboratories) according to the

manufacturer’s instructions and once the reaction was complete, sections were rinsed in PBS.

For the reaction, nickel sulfate was also used. Slides were dehydrated using graded alcohol

solutions and coverslipped using DPX mountant (Electron Microscopy Sciences, Hatfield,

PA, USA).

Quantification of ∆FosB+ cells

The quantification was performed using an Olympus (Center Valley, PA, USA) BX-

51 microscope equipped with a Rotiga 2000R live video camera (QImaging, Surrey, BC,

Canada), a three-axis MAC6000 XYZ motorized stage (Ludl Electronics, Hawthorne, NY,

USA), and a personal computer workstation. The investigators were blind to the treatment

conditions. Contours of the core and shell for the nucleus accumbens and for the dorsal

striatum were drawn at 2X magnification using Stereo Investigator software

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(MicroBrightField, Williston, VT, USA). The grid frame and the counting frame were set to

360 x 360 µm and 75 x 75 µm, respectively. The thickness was measured several times in a

random manner in each animal and an average was used to estimate the total volume of the

sample region and the total number of ∆FosB cells. Two subjects were excluded because

outliers. The results were expressed as the number of ∆FosB-positive cells (cell/volume) for

each of the region studied.

Data and Statistical analyses

Establishment of food addiction-like criteria, food addiction-like score and subpopulations of

rats.

The approach used to establish the food addiction-like criteria and the food addiction-

like score was analogous those previously used to establish addiction-like behaviors in

cocaine self-administering outbred rats (11, 27). Animals were ranked for each criterion

independently. The five-day average of the plateaued intake under FR1 responding, the two-

day average of the breakpoint under the progressive ratio schedule of reinforcement and the

intake in the light/dark conflict test, were used to operationalize the three food addiction-like

behaviors [i) excessive intake, ii) motivation for food, and iii) compulsive-like eating]. The

three food addiction-like criteria were operationally defined based on the following

inclusion/exclusion condition: a subject was considered positive for a given criterion, if it fell

above the 67th

percentile of that distribution. If a rat was considered positive for a given food

addiction-like criterion, it was given an arbitrary criterion score of 1. Then the arbitrary

criteria scores were added so that four different groups were identified depending on the

number of positive criteria they met (from 0 to 3). The food addiction-like score was

calculated as follows (11, 27):

Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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Food Addiction-like score= ∑ ����

where �� = (� − �̅�)/��

The food addiction-like score was calculated as the algebraic sum of the individual

standardized scores(��), of each of the three food addiction-like behaviors (�, excessive

intake, motivation for food, or compulsive-like eating). Each individual standardized score

(��) for a given distribution (�) was calculated by subtracting each behavioral measure (�)

of each individual subject (�) from the mean of that distribution (�̅�) and this difference was

then divided by the standard deviation of that distribution (��). Individual standardized scores

had a mean of 0 and a standard deviation of 1.

Data analysis

Two-way analyses of variance (ANOVAs) with Impulsivity and Food as between-

subjects factors were used to analyze efficiency in the DRL test, Day 1 food intake, food

intake under FR3 and FR5 schedules of reinforcement, breakpoint under a progressive ratio

schedule of reinforcement, intake in the light/dark conflict test, food addiction-like score, and

∆FosB-positive cell counts in the nucleus accumbens core and shell. Changes in daily food

intake over the first 14 days of testing were analyzed using a three-way mixed-design

analysis of variance with Impulsivity and Food as between-subjects factors and Day as a

within-subject factor. To evaluate the internal consistency of measurements and to determine

whether rats stably differed in their individual performances, two-way, random effect

intraclass correlations (ICC) of absolute agreement (9, 13, 17, 28) were performed on the

efficiency during the last 4 days of training in the DRL. Differences in food intake in FR1

schedule of reinforcement, breakpoint under progressive ratio schedule of reinforcement, and

intake in the light/dark conflict test across the 4 criteria were analyzed using one-way

Velázquez-Sánchez et al., High trait impulsivity predicts food addiction-like behavior

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ANOVAs with Criteria as a between-subjects factor. Differences in the food addiction-like

score across the 4 criteria were analyzed using one-way ANOVA with Criteria as a between-

subjects factor. Regression analyses were used to test the relationship between the criteria

and food intake in FR1 schedule of reinforcement, breakpoint under progressive ratio

schedule of reinforcement, and intake in the light/dark conflict test. Pairwise comparisons

were interpreted using Fisher’s LSD tests. The software/graphic packages were Systat 12.0

and SigmaPlot 12.0 (Systat Software Inc., Chicago, IL), SPSS Statistics 18 (SPSS Inc.,

Chicago, IL), InStat 3.0 (GraphPad, San Diego, CA).

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SUPPLEMENTARY FIGURES

Supplementary Figure 1. ∆FosB expression in the dorsal striatum. (A and B) ∆FosB

expression in the dorsal striatum expressed as density change (%) in relation to the Low-

impulsive/Chow group. Statistical analysis revealed a main effect of Food (Food: F1,20=9.5,

p≤0.05). (B) A small correlation between the food addiction-like behavior and ∆FosB

expression in the dorsal striatum was found (r2=0.21, p≤0.05). (C) Representative

micrographs (20X) of ∆FosB expression in dorsal striatum of the different groups are shown.

n=24. Data show M±SEM.

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